Reversible disassembly of the yeast V-ATPase revisited under in vivo conditions

Primary active proton transport by eukaryotic V-ATPases (vacuolar ATPases) is regulated via the reversible disassembly of the V₁V_o holoenzyme into its peripheral catalytic V₁ complex and its membrane-bound proton-translocating V_o complex. This nutrient-dependent phenomenon had been first detected in the midgut epithelium of non-feeding moulting tobacco hornworms (Manduca sexta) and in glucose-deprived yeast cells (Saccharomyces cerevisiae). Since reversible disassembly to date had been investigated mostly in vitro, we wanted to test this phenomenon under in vivo conditions. We used living yeast cells with V-ATPase subunits fused to green, yellow or cyan fluorescent protein and found that only the V₁ subunit C (Vma5) was released into the cytosol after substitution of extracellular glucose with galactose, whereas the other V₁ subunits remained at or near the membrane. FRET analysis demonstrated close proximity between V₁ and V_o even under glucose-starvation conditions. Disassembly, but not reassembly, depended on functional microtubules. Results from overlay blots, pull-down assays and bimolecular fluorescence complementation support the assumption that subunit C interacts directly with microtubules without involvement of linker proteins.